ABSTRACT Interactive keys are tools that aid research and technical work since identification of organisms has become increasingly present in the scientific and academic context. An interactive key was developed with the software Lucid v. 3.3 for the identification of eleven fungal species associated with onion, carrot, pepper and tomato seeds. It was based on a matrix composed of six features: crop, conidium, conidiophore, color of long conidiophore, color of mycelium and presence of setae, besides 21 character states. In addition, descriptions, illustrations and high-resolution photographs of the morphological characters and states were made available to aid in the correct identification of fungal species. Validation of the interactive key was performed by distinct groups of volunteers: (i) graduate students with prior knowledge and using the interactive key; (ii) undergraduate students with little prior knowledge and using the interactive key, and (iii) undergraduate students with little prior knowledge and using the conventional identification system such as the printed manuals used in seed pathology laboratories. We analyzed the time spent by each volunteer to evaluate 25 seeds infected with the fungal species in the key, as well as the percentage of success and the difficulty level for each participant. The high percentage of correct answers with the use of the interactive key and the ease of use by the volunteers confirmed its efficiency because there was an increase in the identification accuracy when compared to the conventional system. Furthermore, the rate of success and the difficulty level presented low variability within groups (i) and (ii). These results are a consequence of the interaction of the user with characteristics of the developed tool, such as high-resolution photographs, which faithfully reproduce the fungal characteristics observed in the seeds under a stereomicroscope. Thus, the interactive key presented here can aid in teaching, institutional and commercial research, inspection and certification of seeds, making diagnosis safer and more accurate. The key is available for free at https://keys.lucidcentral.org/keys/v3/seed_fungi/.