Penicillium species are common fungi found worldwide from diverse substrates, including soil, plant debris, food products and air. Their diversity in aquatic environments is still underexplored. With the aim to explore the fungal diversity in Spanish freshwater sediments, numerous Penicillium strains were isolated using various culture-dependent techniques. A preliminary sequence analysis of the β-tubulin (tub2) gene marker allowed us to identify several interesting species of Penicillium, which were later characterized phylogenetically with the barcodes recommended for species delimitation in the genus. Based on the multi-locus phylogeny of the internal transcribed spacer region (ITS) of the ribosomal DNA, and partial fragments of tub2, calmodulin (cmdA), and the RNA polymerase II largest subunit (rpb2) genes, in combination with phenotypic analyses, five novel species are described. These are P. ausonanum in section Lanata-Divaricata, P. guarroi in sect. Gracilenta, P. irregulare in sect. Canescentia, P. sicoris in sect. Paradoxa and P. submersum in sect. Robsamsonia. The study of several isolates from samples collected in different locations resulted in the reinstatement of P. vaccaeorum into section Citrina. Finally, P. heteromorphum (sect. Exilicaulis) and P. tardochrysogenum (sect. Chrysogena) are reported, previously only known from Antarctica and China, respectively.