Background: Prostate cancer (PCa) is one of the most common malignancies in older men. The study of tissue markers of PCa can provide information about the state of proliferation and apoptosis in tumors, the susceptibility of tumor cells to metastasis and the mechanisms of resistance to therapy, which, in turn, can help predict the course of the disease and develop personalized treatment. Polyamines (PAs) spermine, spermidine, putrescine are of particular interest in terms of PCa tissue markers. Aim: To investigate the levels of basic and acetylated forms of PAs in the postoperative samples of malignant and benign tumors of the human prostate and evaluate the possibility of their use for differential diagnosis and assessment of the PCa aggressiveness. Object and Methods: 57 postoperative tumor samples from patients with prostate adenocarcinoma of different Gleason score (GS) and clinical stage (T1–T4) and 20 samples of tumors from patients with benign prostate hyperplasia (BPH) were studied. The content of PAs was determined by high performance liquid chromatography. Results: Among the studied PAs, the most significant difference between PCa and BPH was observed for spermine (Spm). The level of Spm in PCa samples was 16 times lower than in BPH samples (p < 0.01). We did not find a significant dependence of PAs levels, including Spm, on the clinical stage. The association between the Spm level and the GS was established. The indolent (GS6) tumors were characterized by the highest Spm level while in the most aggressive (GS9 and GS10) tumors Spm content was the lowest. Conclusions: A sharp decrease in Spm levels is probably a characteristic feature of prostate malignant tumors. The obtained results indicate an association of Spm levels in tumors with the GS. This may indicate Spm involvement in the formation of the aggressiveness of PCa. The results of the study can be further used for differential diagnosis of prostate tumors and for assessing the aggressiveness of PCa.