The reproductive system of Orchidaceae is predominantly xenogamous and highly dependent on animal pollen vectors. Nectar is the main floral resource, offered to pollinators in perigonal nectaries, cuniculi or spurs; these structures are often difficult to locate and to evaluate their functionality. The Neotropical Epidendrum denticulatum Barb. Rodr. and Epidendrum orchidiflorum Salzm. ex. Lindl. bloom synchronously throughout the year in the municipality of Maricá (Rio de Janeiro State, SE Brazil). The flowers of E. denticulatum open in the morning, have a conspicuous pink color, a nectar guide, and nuptial and extranuptial nectaries. During the day, they are visited by species of Hesperiidae (Lepidoptera), which remove the pollinia. The flowers of E. orchidiflorum open at night, are green with nuptial and extranuptial nectaries, and have a strong odor at night, suggesting nocturnal pollination agents, possibly moths. To date, no information is available on the reproductive biology and pollination mechanisms for E. orchidiflorum, nor is micromorphological, anatomical or histochemical data related to the presence and functionality of secretory structures associated with pollination in either species. Also, no studies have examined the occurrence of mechanisms that hinder or prevent interspecific pollination. Here, through reproductive, morphological, structural, and histochemical analyses, we evaluated the floral biology and reproductive systems of these synchronopatric Epidendrum species. Both species have extranuptial nectaries, a functional cuniculus, and semaphores. Hand-pollination experiments revealed that the species are self- and incompatible, although individuals with intermediate morphology were not identified. Pre-pollination barriers related to the floral biology of the species seem to be sufficient to prevent hybridization, but other biological aspects certainly contribute to the genetic integrity of the populations, such as the low rates of visits to flowers, low percentage of fruit and seed set, low seedling recruitment, and high investment in vegetative propagation.