In insects, the hemolymph is the primary site of antimicrobial effectors produced in response to infection. In this study, the antibacterial activity of hemolymph extract from immunized larvae of Oryctes owariensis and Rhynchophorus phoenicis has been evaluated. The tests were carried out on in vitro growth of six bacterial strains including three clinical isolates (Escherichia coli ATCC 25922, Bacillus subtilis, Salmonella spp.) and three strains of food origin (Escherichia coli E1, Staphylococcus aureus S1 and S2 Meti-R). Activity of the different extracts was detected by diffusion method in a gel medium and quantified by macro-dilution technique in a liquid medium. Results showed that the different extracts demonstrate strong antibacterial activity and bactericidal power. Diameters of inhibition zones varied from 19.12±2.88 mm (E. coli ATCC 25922) to 22.58±2.18 mm (S. aureus S1) for the extract from O. owariensis infected larvae. This extract was more active on Gram positive bacteria with a MIC of 12.5 mg/mL and a MBC between 25 and 50 mg/mL. The extract from R. phoenicis infected larvae was very active on all strains with inhibition zone diameters between 26.22±0.69 mm (B. subtilis) and 35.30±2, 4 mm (S. aureus S1). MIC and MBC were respectively developed at the concentrations of 3.12 mg/mL and 6.25 mg/mL on all the bacterial strains. The sensitivity of bacterial strains to these hemolymph extracts reveals the probable presence of produced antimicrobial peptides.