Bromelain, a mixture of proteolytic enzymes found in pineapple (Ananas comosus), exhibits antimicrobial properties. This study aimed to evaluate and compare the antimicrobial effects of crude bromelain extracts from different parts of pineapple (crown, core, pulp, and peel) against pathogenic fungi (Mucor mucedo and Aspergillus niger) and bacteria (Staphylococcus aureus and Proteus mirabilis). The extracts were prepared, and their protein content was estimated using Lowry's method. The crown extract had the highest protein concentration (115.83 μg/mL). Bromelain protease activity was determined based on casein hydrolysis and tyrosine release. Antifungal activity was assessed using the disc diffusion method for crown and core extracts, and the agar well diffusion method for pulp and peel extracts. The results showed concentration-dependent inhibition, with higher extract concentrations resulting in larger zones of inhibition against test microorganisms. At 100% concentration, the crown and core extracts exhibited potent antifungal activity against M. mucedo and A. niger, like that of the antifungal drug nystatin. The pulp and peel extracts demonstrated strong antibacterial effects against S. aureus, and the peel extract was slightly more effective. The extracts partially inhibited P. mirabilis. This research highlights the antimicrobial potential of bromelain derived from underutilized parts of pineapple, suggesting its therapeutic application as a natural antimicrobial agent against fungal and bacterial infections.