Bed linen is clearly recognized as a potential reservoir for microorganisms and could be a vector of disease transmission. The present study was aimed at isolating and characterizing bacteria and fungi from different kinds of bed linen of student in Ekiti State University hostels. Pour plate method was used for the enumeration of total bacteria count from the posterior and anterior end of the bed linen. The average bacteria count for the anterior and posterior end was 7.46 and 7.16 Log10 CFU/ml respectively. The most dominant microbial species were bacteria and these were mostly found in the environment and on human skin. The bacteria isolated were Staphylococcus aureus, Staphylococcus epidermidis, Proteus mirabilis, Enterobacter aerogenes, Bacillus cereus, Escherichia coli and Klebsiella aerogenes. Bacillus cereus had the highest frequency of occurrence (25%), followed by Staphylococcus aureus (15%), Escherichia coli (15%), Klebsiella aerogenes (15%), Staphylococcus epidermis (15%), Enterobacter aerogenes (10%) and Proteus mirabilis (5%). The fungi isolates were Aspergillus sulphureus, Aspergillus fumigatus, Aspergillus niger, Aspergillus flavus and Fusarium spp. Antibiotics susceptibility test was carried out on the bacteria isolates with gram negative bacteria showing resistance to Cotrimoxazole and gram positive bacteria showing resistance to Amplicillin. Most of the bacteria isolates have multiple antibiotics resistance. The bacterial isolates were susceptible to Ciprofloxacin. Plasmid profiling was also done with Escherichia coli having three high molecular weight plasmids, Bacillus cereus and Enterobacter aerogenes do not possess plasmid. The identified species are suspected to be opportunistic pathogens for human, representing a risk for people with weakened immune system, especially in cases of super-infection.