We sequenced the genome of Raoultella ornithinolytica strain Marseille-P1025 that caused a rare case of prosthetic joint infection in a 67-year-old immunocompetent male. The 6.7-Mb genome exhibited a genomic island (RoGI) that was unique among R. ornithinolytica strains. RoGI was likely acquired by lateral gene transfer from a member of the Pectobacterium genus and coded for a type IVa secretion system found in other pathogenic bacteria and that may have conferred strain Marseille-P1025 an increased virulence. Strain Marseille-P1025 was also able to infect, multiply within, and kill Acanthamoaeba castellanii amoebae.