Bacterial contamination is a major problem that needs to be addressed in fungal culture. Therefore, this is the first study that successfully eliminated bacterial contamination from aquatic fungal cultures using the Cabin-sequestering (CS) method. To assess its effectiveness, two treatment groups were established: Group A, employing the CS method and Group B, using subculture on antibiotic-free media. The bacteria-fungal mixture, comprising Aspergillus sp. and Aeromonas sp. isolated from the Kuala Ibai mangrove area, was prepared with a ratio of 2:1 (bacteria: fungi). Aeromonas sp. was grown in Tryptic Soy Broth (TSB) and Aspergillus sp. spores on Potato Dextrose Agar (PDA). In Group A, the CS method was applied by piercing a square hole in the growth medium, referred to as a “cabin,” and covering it with a coverslip. After incubation, fungal hyphae that grew beyond the coverslip were transferred onto new PDA plates. This growth was morphologically characterised and confirmed as Aspergillus sp., demonstrating the CS method’s efficacy in ensuring pure fungal growth. In contrast, Group B, which utilised subculturing on antibiotic-free media, showed bacterial growth. This bacteria was streaked on Glutamate Starch Phenol-red (GSP) agar, resulting in yellow colonies that were presumptively identified as Aeromonas sp., highlighting the presence of bacterial contamination in the control group. In conclusion, the CS method, particularly with the 2:1 bacteria-to-fungus ratio, proved to be highly effective in eliminating bacterial contamination and maintaining the purity of fungal cultures. The study’s findings advocate the CS method as a reliable and efficient alternative to traditional subculturing techniques in managing bacterial contamination in fungal cultures. Future research is recommended to explore diverse species and ratios, further validating the CS method’s versatility in different microbial settings.