Free-living amoeba are phagocytic protozoans that act as environmental reservoirs, a protective niche, and a vehicle for transmission for amoeba-resistant bacterial pathogens. Many amoeba-resistant bacteria have been identified using only laboratory-adapted Acanthamoeba. We isolated resident amoeba from target environments of dairy farms and aquaculture settings to evaluate their use as a pathogen detection tool. Amoeba were only isolated from 3 of 23 (13%) environmental samples using established methods. A two-step sample decontamination protocol was developed and led to the isolation of 14 additional amoeba. An amoeba co-culture method was developed to assess the survival of 12 mycobacterial species within environmental and laboratory-adapted amoeba. Major strain differences were observed at the amoeba level which had drastic effects on the survival of different bacterial species within individual amoeba. Targeted isolation of resident bacteria from soils and feces using amoebal enrichment protocols were unsuccessful. However, the methodologies developed in this study provide a valid technical starting point for future studies.