Aims The aims of this study were to identify the structure of antibacterial lipopeptide (LP) produced by Bacillus amyloliquefaciens strain FJAT‐2349, to analyse the effects of the culture medium and temperature on LP production and to assess the biocontrol efficiency of the LPs against tomato bacterial wilt. Methods and Results Lipopeptides were extracted by acid precipitation and resolved in methanol and their structure was identified through LC‐QTOF‐MS/MS method. The antibacterial activities of the LPs were evaluated through inhibition zone experiments. The biocontrol efficiency of the LPs against tomato bacterial wilt was examined by a pot test. The LPs were composed of iturin (C14–C17 iturin A), fengycin (C14/C16 fengycin A, C14 fengycin B2, C16 fengycin A2/B2, C16–C17 fengycin B, C15 fengycin A derivatives and C15 fengycin B derivatives) and surfactin (C12–C16 surfactin A). Moreover, the composition of the LPs was significantly influenced by the culture medium and temperature; the contents of iturin, fengycin and surfactin varied within the range from 0·41–5·89, 4·54–181·67 and 2·05–19·65 mg l−1 in the different culture media and from 0·39–11·04, 1·45–215·14 and 7·79–24·43 mg l−1 under different culture temperatures respectively. The results indicated that the contents of the LP mixture, fengycin and surfactin secreted from FJAT‐2349 all decreased along with an increasing culture temperature. The fermentation supernatants and LP extracts had the strongest inhibition activities of Ralstonia solanacearum when strain FJAT‐2349 was incubated at 25°C using potato dextrose broth as the culture medium among all the assayed culture conditions. The purified fengycin was found to be the active antibacterial compound against R. solanacearum, but the purified surfactin was not. The pot experiments demonstrated that the LPs secreted from the strain FJAT‐2349 could effectively control the tomato bacterial wilt with a biocontrol efficiency of 97·6%. Conclusions The LPs secreted from strain FJAT‐2349 could serve as potential biocontrol agents against tomato bacterial wilt. Significance and Impact of the Study The LPs exhibited good potential applications in the biocontrol of tomato bacterial wilt.