This study identified and sequenced 15 lysin genes from 14 novel virulent phages (10 Gram-negative and 4 Gram-positive bacterial phages). Through heterologous expression in Escherichia coli BL21(DE3), two phage lysins from Vibrio parahaemolyticus, LysCA8 and LysBA3, were obtained and purified, and two phage lysins from Bacillus cereus, LysLY1 and LysLY3, were also obtained without further purification. LysLY1 and LysLY3 had strong lytic activity against all 89 B. cereus strains tested, demonstrating a host range significantly larger than that of their parent phages (vB_BceS_LY1 and vB_BceP_LY3 were only able to lyse 3 and 2 of these B. cereus strains, respectively). LysCA8 and LysBA3 showed no lytic activity against V. parahaemolyticus in vitro, presumably due to environmental acclimation and their single-domain structure. To address this, future work should focus on structural optimization, co-expression of molecular chaperones, and chimeric enzyme design to uncover the inactivation mechanism and expand the antimicrobial spectrum of these lysins.