Pediculosis capitis caused by Pediculus humanus capitis (De Geer) is endemic all over the world, and children are mostly affected, particularly those living in overcrowded institutions. Several studies have shown that P. h. capitis carried human pathogenic bacteria, suggesting the potential role of head lice in the transmission of pathogens to humans. In this study, we determined the genetic diversity of head lice collected from welfare homes sheltering underprivileged children by using DNA barcoding and demonstrated the presence of Acinetobacter spp., Serratia marcescens, and Staphylococcus aureus in head lice, which have never been investigated before in Malaysia. Cox1 DNA barcoding identified the head lice, P. h. capitis collected from welfare homes across two geographical areas of Peninsular Malaysia as belonging to clades A, B, and D. Acinetobacter bacteria: Acinetobacter guillouiae, Acinetobacter junii, Acinetobacter baumannii, and Acinetobacter nosocomialis were detected in head lice belonging to clades A and also D. In addition, DNA from S. marcescens and S. aureus were also detected in both clades A and D. To our knowledge, this is the first report on the genetic diversity of head lice in Malaysia through DNA barcoding, as well as the first to provide molecular evidence on the type of bacteria occurring in head lice in Malaysia. It is anticipated that the DNA barcoding technique used in this study will be able to provide rapid and accurate identification of arthropods, in particular, medically important ectoparasites.