A bryophyte diversity survey was carried out from July 29 to 1 August 2013, in Santa Cruz das Flores, Flores Island (Azores) (39.471185 N Latitude; -31.184692 W Longitude), along an elevational gradient (70, 200, 400, 600 and 800 m a.s.l.). The study employed the Global Island Monitoring Scheme (GIMS) protocol for bryophytes. At each elevation level, three replicates of six substrates colonised by bryophytes (rock, soil, humus, dead wood, tree trunks, leaves) were collected. In total, 385 sampling events generated 1345 species occurrence records, representing 89 bryophyte species (37 mosses; 52 liverworts). The acrocarpous moss Fissidens azoricus (P.de la Varde) Bizot, listed as "Critically Endangered" by the IUCN, had not been reported from Flores Island since 1937; this is the first publication with new locations for the species. The altitudinal gradient revealed an increase in species richness and abundance with elevation, following a mid-elevation peak pattern, with the highest richness between 400 m a.s.l. (52 species; 364 records) and 600 m a.s.l. (54 species; 402 records), followed by a decline at 800 m a.s.l. (33 species; 148 records). At 70 m a.s.l., 20 species were identified (128 records) and, at 200 m a.s.l., 35 species were recorded (232 records). In terms of substrate preference, the highest species richness and abundance were found colonising epiphytic substrates (58 species; 424 records), followed by terricolous substrates (44 species; 233 records).