Background Acute kidney injury (AKI) is a worldwide public health problem and is often treated by non-nephrologists. The objective of this study was to evaluate the knowledge of non-nephrologists, undergraduate medical students and health professionals regarding AKI. Methods An unsupervised and closed-response electronic questionnaire was sent to sixth-year medical students and non-nephrologist health professionals working in the city of São Paulo, Brazil. Results A total of 424 responses were returned from 650 invitations (40.1% males, 39.2% physicians, 34.0% senior medical students or resident physicians, 16.3% nurses and 10.6% pharmacists). The knowledge of medical students and health professionals about the prevalence and lethality of AKI in hospitals ranged from 40.8% to 59.2%. The most recognized susceptibilities and risk factors for AKI were sepsis/septic shock (95.0%) and diabetes mellitus (91.3%); the less-recognized susceptibilities and risk factors were exposure to gadolinium-based contrast (23.3%) and chronic liver disease (55.7%). The study participants’ rate of knowledge regarding the diagnosis of AKI was 50.9–73.6%, and their rate of knowledge regarding the criteria of increased serum creatinine and reduced urine volume was 52.6%. The most-recognized nephrotoxic medications were vancomycin (82.3%) and diclofenac (80.4%), and the least-recognized were acyclovir (34.0%) and cotrimoxazole (30.4%). The indications for emergency renal replacement therapy that were recognized by the study respondents were metabolic acidosis (82.3%), uremic syndrome (81.6%), hyperkalemia unresponsive to clinical treatment (78.1%) and acute pulmonary edema (71.6%). Conclusion The study showed knowledge gaps that can impact patient care and can be used to guide professional training programs.