(1) Background: Little is known about the impact of urinary microflora, in particular, its effects on side effects after radiotherapy. The use of mass spectrometry identification method (MALDI) may bring a new look at the issue of the composition and significance of the urinary microbiome. This study aimed to use the mass spectrometry identification method (MALDI) to identify the microbiome of urine samples collected from 50 irradiated prostate cancer patients. (2) Methods: Blood and urine samples were collected before gold marker implantation, at the start and last day of radiotherapy, 1, 4 months after. Patients do not always collect the urine from the midstream; therefore, samples were collected from the first void and midstream in 12 patients for MALDI analysis; in the remaining 38 patients—from the midstream void for MALDI and biochemical analysis. (3) Results: Microorganisms were present in 140/181 urine samples. We found 33 different species 3G(−) and 30G(+). The most frequently isolated strains were: Staphylococcus haemolyticus, Staphylococcus epidermidis, Staphylococcus hominis, Enterococcus faecalis, and Micrococcus luteus. When comparing the type of urine samples, bacteria were more common in samples from the first-void urine than from the midstream one. The absence of bacteria was found in 12.2% of samples from the first-void urine and in 24.7% from the midstream. There was no difference in the total incidence of species between streams (p = 0.85). Before fiducial implantation, the total number of detected bacterial species was significantly higher in comparison to the end of radiotherapy (p = 0.038), indicating that the administered therapy resulted in depleting the local microbiome. One month after radiotherapy, an increase in the number of isolated bacteria was observed. The number of bacterial species in urine did not correlate with blood parameters. The presence of leukocytes (p = 0.013) and proteins (p = 0.004) in urine was related to a greater variety of bacteria found in urine specimens. (4) Conclusions: We obtained a similar spectrum of bacteria from the initial and middle urine streams. We also showed that there is a change in bacteria species affected by the treatment of prostate cancer patients, with both antibiotics before gold fiducial implantation and radiotherapy.