Endophytic bacteria associated with Opuntia ficus-indica play a crucial role in the plant’s immune defense mechanisms against biotic stress, particularly pest infestations. This study investigates the impact of cochineal infestation (Dactylopius opuntiae) on the diversity and ecological roles of endophytic bacteria associated with Opuntia ficus-indica in the semi-arid region of Beni Mellal, Morocco. Healthy and infested cactus cladodes were sampled, and endophytic bacteria were isolated using selective media. Bacterial identification involved morphological characterization, biochemical profiling, and molecular analysis through 16S rRNA sequencing. Thermal adaptability tests and statistical analyses (one-way ANOVA, Tukey’s test) were conducted to assess strain adaptability and ecological significance. Of the 57 bacterial isolates obtained, nine dominant strains were selected based on colony abundance (>105 CFU/mL), distinct biochemical profiles, and ecological relevance. Healthy cactus tissues demonstrated significantly higher endophytic colonization (mean = 6.5 isolates per sample) compared to infested tissues (mean = 3.0 isolates; ANOVA, F(1,10) = 8.64, p = 0.014). Bacillus cereus (Mg1C) and Pseudomonas aeruginosa (Mg1F) predominated in healthy cladodes, suggesting potential roles in pest resistance or plant defense. Conversely, Proteus penneri (Mg1H), typically associated with insect guts and tissue decomposition, dominated in infested tissues. Thermal tolerance assays revealed significant adaptability differences among isolates (ANOVA, F(8,27) = 11.87, p < 0.001), notably highlighting P. aeruginosa, P. putida, and Klebsiella pneumoniae as highly thermotolerant strains with promising biocontrol potential. The geographic scope of this study was limited, and laboratory conditions may not entirely replicate complex field interactions. Nevertheless, the identified bacterial strains exhibit strong potential as bioinoculants in sustainable agriculture for pest management and enhancing plant resilience under stress conditions. This research uniquely elucidates the impact of pest infestation on cactus-associated endophytic communities, offering valuable insights into microbial ecology and practical agricultural management strategies.