Objective To investigate the expression and significance of human neutrophil lipocalin (HNL) and neutrophil CD64 (nCD64) in prostatic fluid of chronic prostatitis patients with different infection degrees. Methods One hundred and seventy-eight patients with chronic prostatitis admitted to the Affiliated Ethnic Hospital of Guangxi Medical University from February 2017 to October 2019 were selected as the research objects. According to whether they were infected with bacteria, the patients were divided into chronic prostatitis/chronic pelvic pain syndromes group (CP/CPPS group, n=40) and chronic bacterial prostatitis group (CBP group, n=138). According to National Institutes of Health Chronic Prostatitis Symptom Index (NIH-CPSI)score, CBP group was divided into mild group (n=65), moderate group (n=46) and severe group (n=27), and another 40 healthy people were selected as control group. Age, body mass index (BMI), course of disease, white blood cell (WBC), and NIH-CPSI score were recorded and analyzed. The expression level of HNL in prostatic fluid was detected by enzyme linked immunosorbent assay (ELISA). The expression level of nCD64 in prostatic fluid was detected by flow cytometry. Pearson correlation was used to analyze the correlation between both HNL and NCD64 levels and NIH-CPSI score in the CBP group. Logistic regression analysis was used to analyze the influencing factors of CBP and CP/CPPS. ROC curve was used to evaluate the diagnostic value of HNL and nCD64 for CBP. Results Compared with the control group, WBC, HNL and nCD64 levels in CP/CPPS group, CBP group and its subgroups were significantly increased (P<0.05), while the CBP group was higher than CP/CPPS group, and the CBP subgroup increased with the increase of infection degree, and the difference between the groups was statistically significant (P<0.05). There was no significant difference in NIH-CPSI score between CP/CPPS group and CBP group (P>0.05), but NIH-CPSI score of moderate group was higher than that of mild group, and that of severe group was higher than that of moderate group and mild group(P<0.05). Pearson correlation analysis showed that the levels of HNL and nCD64 in prostatic fluid of CBP group were significantly positively correlated with NIH-CPSI score (r=0.897, P<0.001; r=0.919, P<0.001). Multivariate logistic regression analysis showed that bacterial infection, HNL, and nCD64 were independent risk factors for CBP (P<0.05). Other pathogen infections, urinary dysfunction, neuroendocrine abnormalities, abnormal immune response, and pelvis related diseases were independent risk factors for CP/CPPS (P<0.05). ROC curve analysis showed that HNL and nCD64 in prostatic fluid had higher diagnostic value for the occurrence of CBP, area under the ROC curve (AUC) was 0.837 and 0.899 respectively, and the best critical values were 165.36 μg/L and 4078.28/cells respectively. The combination of the two (HNL and nCD64) had higher diagnostic value, with AUC of 0.949. Conclusion The levels of HNL and nCD64 in prostatic fluid of patients with chronic prostatitis increased with the aggravation of the bacterial infection, showing a positive correlation, and the combined monitoring of HNL and nCD64 is of reference value for clinical diagnosis and the judgment of infection degree.