The purpose of this study was to establish an infection model of Galleria mellonella larvae as an alternative in vivo model for biofilm-associated infections on stainless steel and titanium implants. First, the model was established with sterile implants to evaluate biocompatibility. Titanium or stainless steel implants were implanted without adverse effects over the entire observation period of 5 days compared to controls and even up to the pupae and moth stage. Then, stainless steel and titanium implants contaminated with Staphylococcus aureus were implanted into larvae to mimic biofilm-associated infection. For both materials, pre-incubation of the implant with S. aureus led to significantly reduced survival of the larvae compared to sterile implants. Larvae could not be rescued by gentamicin, whereas gentamicin significantly improved the survival of the larvae in case of planktonic infection with S. aureus without an implant, confirming the typical characteristics of reduced antibiotic susceptibility of biofilm infections. Biofilm formation and various stages of biofilm maturation were confirmed by surface electron microscopy and by measuring bacterial gene expression of biofilm-related genes on contaminated implants, which confirmed biofilm formation and upregulation of autolysin (atl) and sarA genes. In conclusion, G. mellonella can be used as an alternative in vivo model to study biofilm-associated infections on stainless steel and titanium implants, which may help to reduce animal infection experiments with vertebrates in the future.