The diversity and antimicrobial activities of 80 fungi isolated from Haliclona simulans were assessed using different fungal media containing either agar or gellum gum. In total, 19 different genotypes were detected. These fungal isolates could be classified as members of the Agaricomycotina, Mucoromycotina, Saccharomycotina, and Pezizomycotina, although the majority of the isolates were associated with the latter class. Some of these fungal isolates showed antimicrobial inhibition of Escherichia coli, Bacillus sp., Staphylococcus aureus, and Candida glabrata. Fungal 18S rRNA gene sequences belonging to Eurotiales, Calosphaeriales, and Chaetothyriales were amplified from DNA and RNA extracted from this marine sponge. This study indicates that in contrast to the low diversity of fungi detected by polymerase chain reaction (PCR) and reverse transcription (RT)-PCR amplification from extracts of this marine sponge, a much higher diversity of fungi could be cultured. The data suggests that some fungi live in symbiosis with H. simulans, whereas other fungi may have been ingested from the surrounding seawater.