Pattern-triggered immunity (PTI) is activated in plants upon recognition by pattern recognition receptors (PRRs) of damage- and microbe-associated molecular patterns (DAMPs and MAMPs) derived from plants or microorganisms, respectively. To understand better the plant mechanisms involved in the perception of carbohydrate-based structures recognized as DAMPs/MAMPs, we have studied the ability of mixed-linked β-1,3/1,4-glucans (MLGs), present in some plant and microbial cell walls, to trigger immune responses and disease resistance in plants. A range of MLG structures were tested for their capacity to induce PTI hallmarks, such as cytoplasmic Ca2+ elevations, reactive oxygen species production, phosphorylation of mitogen-activated protein kinases and gene transcriptional reprogramming. These analyses revealed that MLG oligosaccharides are perceived by Arabidopsis thaliana and identified a trisaccharide, β-d-cellobiosyl-(1,3)-β-d-glucose (MLG43), as the smallest MLG structure triggering strong PTI responses. These MLG43-mediated PTI responses are partially dependent on LysM PRRs CERK1, LYK4 and LYK5, as they were weaker in cerk1 and lyk4 lyk5 mutants than in wild-type plants. Cross-elicitation experiments between MLG43 and the carbohydrate MAMP chitohexaose [β-1,4-d-(GlcNAc)6], which is also perceived by these LysM PRRs, indicated that the mechanism of MLG43 recognition could differ from that of chitohexaose, which is fully impaired in cerk1 and lyk4 lyk5 plants. MLG43 treatment confers enhanced disease resistance in A. thaliana to the oomycete Hyaloperonospora arabidopsidis and in tomato and pepper to different bacterial and fungal pathogens. Our data support the classification of MLGs as a group of carbohydrate-based molecular patterns that are perceived by plants and trigger immune responses and disease resistance.