Antibiotics　and　herbicides　are　contaminants　of　emerging　concern　in　aquatic　environments.　Lake　Villarrica　is　a　relevant　freshwater　body　in　Chile　and　was　recently　designated　a　‘saturated　nutrient　zone’.　Here,　we　investigated　the　occurrence　of　multiple　antibiotic　resistance　(MAR)　and　herbicide　catabolic　profiles　among　bacteria　present　in　the　surface　sediments　of　Lake　Villarrica.　The　occurrence　of　antibiotic-resistant　genes　(ARGs;　blaTEM,　catA　and　tetM)　and　herbicide-catabolic　genes　(HCGs;　phnJ　and　atzA)　was　investigated　by　qPCR.　Subsequently,　the　presence　of　culturable　bacteria　with　multiple　resistance　to　amoxicillin　(AMX),　chloramphenicol　(CHL)　and　oxytetracycline　(OXT)　was　studied.　Forty-six　culturable　MAR　(AMX　+　CHL　+　OXT)　strains　were　isolated　and　characterized　with　respect　to　their　resistance　to　11　antibiotics　by　using　a　disc　diffusion　assay　and　testing　their　ability　to　use　herbicides　as　a　nutrient　source.　qPCR　analyses　revealed　that　ARGs　and　HCGs　were　present　in　all　sediment　samples　(101　to　103　gene　copies　g−1),　with　significant　(P　≤　0.05)　higher　values　in　sites　near　Villarrica　city　and　cattle　pastures.　The　plate　method　was　used　to　recover　MAR　isolates　from　sediment　(103–106　CFU　g−1),　and　most　of　the　46　isolates　also　showed　resistance　to　oxacillin　(100%),　cefotaxime　(83%),　erythromycin　(96%)　and　vancomycin　(93%).　Additionally,　54　and　57%　of　the　MAR　isolates　were　able　to　grow　on　agar　supplemented　(50　mg　L−1)　with　atrazine　and　glyphosate　as　nutrient　sources,　respectively.　Most　of　the　MAR　isolates　were　taxonomically　close　to　Pseudomonas　(76.1%)　and　Pantoea　(17.4%),　particularly　those　isolated　from　urbanized　sites　(Pucón　city).　This　study　shows　the　presence　of　MAR　bacteria　with　herbicide　catabolic　activity　in　sediments,　which　is　valuable　for　conservation　strategies　and　risk　assessments　of　Lake　Villarrica.　However,　major　integrative　studies　on　sediments　as　reservoirs　or　on　the　fate　of　MAR　strains　and　traces　of　antibiotics　and　herbicides　as　a　result　of　anthropic　pressure　are　still　needed.　©　2024　Elsevier　Ltd