Summary Background Sporotrichosis is a group of zoonotic subcutaneous mycoses, found worldwide and caused by fungi belonging to the genus Sporothrix. Protozoans of the genus Acanthamoeba are widely distributed, and some species may be pathogenic and/or opportunistic. These organisms coexist in the same environment and may interact. Objectives This study determined the profile of interactions of S schenckii sensu stricto and S brasiliensis with A castellanii, using an in vitro co‐culture model to evaluate the intrinsic characteristics of the two Sporothrix species and A castellanii. Methods We compared the rate of phagocytosis of S schenckii sensu stricto and S brasiliensis by A castellanii; the viability of S schenckii sensu stricto and S brasiliensis after contact with A castellanii; the viability of the amoeba after contact with a fungal species; and the influence of S schenckii sensu stricto and S brasiliensis on the encystment process of A castellanii. Results The analyses indicated that A castellanii phagocytised both S schenckii and S brasiliensis, with significantly more S schenckii than S brasiliensis in the first two hours of contact. Our results showed a significant increase in conidia and hyphae count after 72 hours of co‐culture of A castellanii with S brasiliensis, and the amoebae lysed after they ingested the fungi, indicating that the fungi probably used the amoebae as a source of nutrition. Conclusions Our results were obtained in vitro and these organisms may not behave similarly in vivo; in vivo studies of co‐infections are necessary in order to gain a thorough understanding of this relationship.