This work aimed to assess the diversity and microhabitat use of benthic invertebrates in an urban forest stream in southeastern Brazilian region. The invertebrates were sampled in a headwater stream, located at the Tijuca Forest, Rio de Janeiro. Three types of microhabitats were sampled (litter, sand and stone) using a Surber sampler. Specimens were identified to the family-level and rarefaction curves were constructed for the total sampling and for each type of microhabitat. Community structure indices (abundance, taxonomic richness, diversity, and dominance) were calculated for each microhabitat. Differences among indices were tested through a repeated measure ANOVA, and differences among microhabitatsʼ composition through a PerMANOVA. A total of 9,800 specimens were registered in which Chironomidae was the most abundant. The rarefaction curves did not reach the asymptote. Community structure indices exhibited differences (RM ANOVA; p < 0.001), as well as microhabitats’ composition (PerMANOVA; p < 0.001). Abundance and taxonomic richness were the highest in litter, diversity was higher both in litter and stone, and sand had the highest dominance. Results highlighted that the variety of microhabitats may enhance local diversity and that the differences in resources availability of each type of microhabitat determine the distribution of these invertebrates.