The effect of Wolbachia on the viability and antimicrobial activity of the ectoparasitoid Habrobracon hebetor was evaluated in laboratory experiments. Two lines of the parasitoid, Wolbachia-infected (W+) and Wolbachia-free (W−), were used. Parasitoid larvae were fed with a host orally infected with a sublethal dose of Bacillus thuringiensis (Bt) and on the host uninfected with Bt. Parasitoid survival was assessed at developmental stages from second-instar larvae to adults. At all developmental stages, there were no statistically significant differences in survival between lines W+ and W−, regardless of host Bt infection. In both W+ and W− lines, the expression of lysozyme-like proteins, antimicrobial peptides (AMPs), and Hsp70 genes was analysed in fourth-instar larvae fed with an infected and uninfected host. In addition, lysozyme-like activity and antibacterial activity were evaluated. The expression of AMPs was significantly higher in W− larvae and did not get induced during the feeding on the Bt-infected host. mRNA expression of lysozyme-like proteins and lysozyme activity were significantly higher in W+ larvae than in W− larvae and did not get induced when the larvae were fed with the infected host. In whole-body homogenates of H. hebetor larvae fed with the uninfected host, antibacterial activity against gram-positive bacteria (Bacillus cereus and Bacillus subtilis) was significantly higher in the W+ line and did not get induced during the feeding with the Bt-infected host. Therefore, there is no obvious immunostimulatory effect of Wolbachia in H. hebetor larvae when they feed on a host infected with an entomopathogenic bacterium.