We investigated the resistance genes, pilus islets, biofilm formation ability and sequence types of multidrug-resistant
Streptococcus pneumoniae
(MDRSP) isolated from healthy children below 5 years of age in Indonesia. In all, 104 archived MDRSP isolates from previous carriage studies in Indonesia in 2016–2019 were screened for the presence of antibiotic resistance genes and the rrgC (pilus islet 1) and pitB (pilus islet 2) genes. Multilocus sequence typing and biofilm formation were determined by PCR sequencing and the ability of cells to adhere to the walls, respectively. Results have shown that the mefA, ermB and tetM genes were found in 93, 52 and 100 % of MDRSP isolates, respectively. Insertions of arginine, proline and Ile-100–Leu were the most common mutations in the folA and folP genes. Pilus islets 1 and 2 were discovered in 93 and 82 % of MDRSP isolates, respectively. The MDRSP isolates showed no biofilm formation ability (64 %), and 5 out of 10 strains of MDRSP strains were ST1464. This finding can be used to provide further considerations in implementing and monitoring pneumococcal vaccination in Indonesia.