Pharmaceutical oral dosage forms intended to children have to be administered under liquid dosage forms. These oral liquid formulations contained into multidose containers require the addition of antimicrobial agents to avoid the growth of microorganisms. Moreover, the oral administration of poorly water soluble drugs requires the use of excipients (organic solvent, cyclodextrins, surfactants, polymers) able to improve their water solubility. 2-pyrrolidone (Soluphor ® P) a co-solvent usually used to dissolve drugs for the manufacture of parenteral dosage forms and suggested also for other administration routes, showed antimicrobial activity. To study the extent of its preservative efficacy, we determined the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) first, for bacteria (Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Enterococcus faecalis, Staphylococcus epidermidis, Staphylococcus capitis) and for bacterial spores (Bacillus cereus, Bacillus sphaericus, Bacillus subtilis), then for yeast and mould (Candida albicans, Aspergillus niger). The results showed that 2-pyrrolidone has a bactericidal and fungistatic efficacy. Challenge tests were realized on oral aqueous paediatric formulations of vitamin D3 containing 2-pyrrolidone as a co-solvent and all known micro-organisms in such formulations were studied. The preservative efficacy of 2-pyrrolidone was observed at a dosage level of 75 mg/mL.