Quality seeds of improved local genotypes is an important input for increasing the productivity of groundnut in Ghana. The existing means of meeting groundnut seed requirements, especially by smallholder farmers, have serious challenges with timely supply and access to these improved genotypes as a result of the limited participation of the private sector and the self-pollinated nature of the crop. Smallholder farmers who take the initiative to store their own seeds, have challenges with storage fungi and aflatoxin contamination. Farmers’ groundnut seed stocks have shown that improvement in seed quality and farmers’ seed management requires maintaining healthy seed stock. Toxicogenic fungi and mycotoxins have been reported in several human and animal health disorders and are major contaminants of groundnut seeds during storage. Some level of success has, however, been achieved from earlier studies to evaluate the efficacy of some plant botanicals for preserving shelled groundnuts. However, for smallholder on-farm safety, such phyto-based preservation methods rather reduce groundnut seed embryo vigour and germination rates. Designing and developing economically appropriate storage solution (practices) for maintaining seed integrity for use requires evidence-based research and an eco-friendly approach. Thus, this study was aimed at assessing the efficacy of using jute bag (JB) and interlaced polyethylene jute bag (IPJB) combinations for the storage of groundnut seeds (varieties) against fungal infection and aflatoxin contamination under ambient storage conditions. The study was undertaken at the Department of Biochemistry, Kwame Nkrumah University of Science and Technology (KNUST), Kumasi, Ghana. Seeds were packaged and stored either in jute bags (JB) or interlaced polyethylene jute bags (IPJB) at ambient conditions over four months’ period. All seeds of groundnut varieties were partly colonised by a range of Aspergillus, Fusarium and Rhizopus spp of fungi. Aflatoxins were detected in only Nkosour (148.21 ppb) while Adepa and Kwame Danso recorded elevated levels of aflatoxin B1 (45.918 ppb) and B2 (410.974 ppb) at four months after storage (MAS). Results indicate that, the IPJB packaging was effective for short-term storage only, while the level of pathogen infection and aflatoxin contamination recorded was low. However, none of the packages evaluated proved exceptionally efficient. Irrespective of the storage package used, Mireku, Konkoma, Nkate Broni, Kumawu Local, Shitaochi, Azizivi, and Jenkaah recorded biologically and economically insignificant levels of aflatoxins. Thus, planting these groundnut varieties by smallholder farmers may offer them some level of security from aflatoxin contamination and loss of seed quality.