As aquaculture expands globally, understanding immune responses in non-traditional farmed species like Octopus vulgaris under varying environmental conditions is increasingly important. This study investigated lysozyme activity, a key innate immune marker, in cell-free hemolymph of O. vulgaris following experimental challenge with four Gram-negative fish pathogens (Photobacterium damselae subsp. piscicida, P. damselae subsp. damselae, Vibrio alginolyticus, and V. anguillarum O1) at two temperatures (21 ± 0.5 °C and 24 ± 0.5 °C). These pathogens were selected because octopus farming frequently occurs near fish aquaculture facilities, raising the potential for pathogen crossover. A total of 216 wild octopuses were injected intramuscularly or intravenously and sampled on days 0, 3, and 7 post-challenge. Lysozyme activity varied by pathogen, injection route, sampling time, and temperature. A significant time- and temperature-dependent increase was observed, especially in IM-challenged groups exposed to Photobacterium species. Elevated temperatures supported a more prolonged immune response. These results highlight lysozyme as a responsive biomarker of innate immunity in O. vulgaris and emphasize the role of environmental factors in immune modulation. This work provides a foundation for disease monitoring and health management in cephalopod aquaculture. Future research should examine long-term lysozyme dynamics, broader pathogen exposure, molecular mechanisms, and additional environmental stressors such as salinity and pollution.