The current literature does not support the usefulness of clinical markers on predicting which patients with atypical small acinar proliferation (ASAP) are more likely to progress to prostate cancer (PCa). Androgens have long been considered to be the potential risk factors for PCa. However, the role of testosterone is controversial. The present study aims to analyze the relationship between serum testosterone (TS) levels and the diagnosis of PCa after a first prostate biopsy in patients affected by ASAP. This retrospective study included 143 patients diagnosed with ASAP in an initial transrectal ultrasound-guided prostate biopsy for suspicious PCa according to the European Association of Urology guidelines. Their TS levels, age, PSA, prostate volume, digital rectal examination, and prostate biopsy Gleason score (GS) were collected retrospectively for statistical analysis. All patients included in the study had a second biopsy and were suitable for further analysis. Re-biopsy was carried out 3–6 months after the first diagnosis of ASAP. Low and normal TS groups were composed of 29 (20.3%) and 114 (79.7%) patients, respectively. The diagnosis of the second biopsy was ASAP in 25.2% and PCa in 36.4% of patients. The comparison between patients with PCa and those with negative or an ASAP result in the second biopsy reported that men with cancer had significantly higher levels of TS (P < 0.001). However, there was no statistically significant association between GS postbiopsy and TS (P = 0.324). Our experience demonstrated that eugonadal patients may be a clinical risk factor for the diagnosis of PCa on re-biopsy after ASAP diagnosis than hypogonadal.