Some insects display immunological priming as a result of elevated humoral and cellular responses which give enhanced survival against subsequent infection. The humoral immune response of Galleria mellonella larvae following pre-exposure to heat killed Staphylococcus aureus or Candida albicans cells was determined by quantitative mass spectrometry in order to assess the relationship between the humoral immune response and resistance to subsequent bacterial or fungal infection. Larvae pre-exposed to heat killed S. aureus showed increased resistance to subsequent bacterial and fungal infection. Larvae displayed an increased hemocyte density (14.08 ± 2.14 × 106 larva− 1 (p < 0.05) compared to the PBS injected control [10.41 ± 1.67 × 106 larva− 1 ]) and increased abundance of antimicrobial proteins (cecropin-D-like peptide (+22.23 fold), hdd11 (+12.61 fold) and prophenol oxidase activating enzyme 3 (+5.96 fold) in response to heat killed S. aureus. Larvae pre-exposed to heat killed C. albicans cells were resistant to subsequent fungal infection but not bacterial infection and showed a reduced hemocyte density (6.01 ± 1.63 × 106 larva− 1 (p < 0.01) and increased abundance of hdd11 (+32.73 fold) and moricin-like peptide C1 (+16.76 fold). While immune priming is well recognised in G. mellonella larvae the results presented here indicate distinct differences in the response of larvae following exposure to heat killed bacterial and fungal cells.