Background Fentanyl has led to an increased number of overdose deaths in North America. Testing substances for fentanyl may be a harm reduction strategy to prevent overdose. Little is known about behavior change after fentanyl testing and the attitudes around fentanyl knowledge and testing along the US-Mexico border in the context of a safe consumption site. Methods This was a pilot quantitative and qualitative study with 30 women who use drugs at an unsanctioned safe consumption site in Mexicali, Mexico. Women participated in a quantitative survey, a semi-structured interview, and fentanyl testing of substances. Injection behavior was observed after fentanyl testing results were provided. Qualitative data were collected to explore the meanings participants attributed to fentanyl and fentanyl testing. Results Half of the substances tested positive for fentanyl (n=15, 50%), and all of them were in samples of black tar heroin. Among those participants who tested positive for fentanyl, 7 (47%) subsequently used less of the intended substance, 1 did not use the intended substance, and 7 (47%) did not change their behavior (i.e., used as originally intended). In qualitative interviews, a predominant theme was a description of fentanyl as dangerous and deadly and fentanyl testing as being helpful for modifying drug use behaviors. However, participants recognized that there could be no change in behavior following a positive fentanyl test in the context of not being able to find substances free of fentanyl. Conclusion We observed mixed results related to behavior change after women's intended substance for use tested positive for fentanyl. Fentanyl testing was acceptable to women, but behavior change was hampered by the inability to find substances free of fentanyl. Further research is needed to maximize the potential of fentanyl testing as a harm reduction tool especially in the context of a changing drug supply.