Phospholipases A₂ (PLA₂s) are key mediators of the cytotoxic and inflammatory activities of snake venoms. While PLA₂ isoforms from Bothrops diporus venom have been characterized and shown to possess antimetastatic and antiangiogenic properties, their impact on mitochondrial bioenergetics and immune modulation has not yet been investigated. In this study, we examined the bioenergetic and immunomodulatory effects of B. diporus PLA₂s using integrated biochemical, metabolic, and multiplex cytokine analyses. In MDA-MB-231 breast cancer cells, pooled PLA₂s induced a dose-dependent decrease in MTT-reducing activity, increased mitochondrial ROS, caused Δψm hyperpolarization, and decreased NADH autofluorescence, collectively indicating sustained mitochondrial stress. Real-time impedance measurements further revealed a marked inhibition of cell proliferation. In human PBMCs, pooled PLA₂s elicited a dynamic cytokine program, inducing early cytotoxic (Granzyme B) and chemotactic (CCL2, CCL3, CCL4) mediators, followed by late pro-inflammatory and regulatory factors such as IL-6, TNF-β, IL-10 and IL-15. Analysis of a single purified PLA₂ isoform (Fraction 6) confirmed activation of the canonical IL-6/TNF-α/IL-1β axis but uniquely induced IL-10 and CCL20, revealing isoform-specific immunomodulatory properties. Altogether, these findings provide the first integrated characterization of mitochondrial and immune perturbations induced by B. diporus PLA₂s, expanding their recognized biological scope and underscoring their potential as molecular templates for novel pharmacological strategies targeting mitochondrial vulnerabilities or modulating the tumor immune microenvironment.