Prostatitis is an inflammatory disorder of the prostate gland, commonly associated with pelvic discomfort, urinary disturbances, and, in some cases, systemic symptoms such as fever. Bromelain, a proteolytic enzyme complex derived from Ananas comosus, is widely recognized for its broad range of beneficial effects. Phytosterols, largely employed to maintain prostate well-being, may exert beneficial actions by modulating lipid metabolism and inflammatory pathways. This study aimed to investigate the anti-inflammatory activity of bromelain (1850 GDU/g) and phytosterols, individually and in combination, using an ex vivo model of lipopolysaccharide (LPS)-induced prostatitis in mouse prostate tissues. In accordance with the principles of “Replacement, Refinement and Reduction in Animals in Research”, the study protocol was approved by the local ethical committee (‘G. d’Annunzio’ University, Chieti, Italy) and Italian Health Ministry (Project no. F4738.N.ZDZ ). The cytotoxicity of the two components (1, 5, 10, 20, 40 mg/ml) was also evaluated in human fibroblast cells (HFF-1) using the MTT assay. Three combinations were analyzed: mixture-1 (bromelain 500 µg/mL + phytosterols 860 µg/mL), mixture-2 (bromelain 5 mg/mL + phytosterols 8.6 mg/mL), and mixture-3 (bromelain 50 mg/mL + phytosterols 86 mg/mL). Gene expression of pro-inflammatory markers, including cyclooxygenase-2 (COX-2), nuclear factor-κB (NF-κB), and tumor necrosis factor-α (TNF-α), was evaluated in prostate tissues via Real-Time PCR. The tested compounds exhibited no cytotoxic effects, with cell viability values remaining above 90% at all concentrations. Mixture-1 selectively reduced TNF-α expression, while mixtures 2 and 3 showed broader anti-inflammatory activity, downregulating COX-2, NF-κB, and TNF-α. In summary, this study provides the first ex vivo evidence of a synergistic anti-inflammatory effect of bromelain and phytosterols in inflamed prostate tissue. These results support the potential of this plant-based formulation as a complementary approach in the management of prostatitis. Further studies are warranted to confirm these findings and elucidate the underlying mechanisms.