Abstract Objective To assess the real-world effectiveness of targeting biologic DMARDs (bDMARDs) in rheumatoid arthritis (RA) patients negative for rheumatoid factor (RF) and anti-citrullinated protein antibodies (ACPA). Methods We retrospectively selected 81 seronegative and 404 seropositive RA patients receiving treatment with abatacept, anti-TNF alpha or tocilizumab. Effectiveness was evaluated by analysing drug survival using Kaplan–Meyer analysis over 10-year follow-up. Survival rates were compared by log rank test, and hazard ratios (HRs) of therapy discontinuation were estimated through multivariate Cox regression. Results Clinical characteristics were similar between the two groups, except for a significantly higher percentage of inadequate responders to prior bDMARDs in the seronegative RA patients (P = 0.02). Among seronegative RA, tocilizumab demonstrated a survival rate of 73.9% with a mean survival time (MST) of 76.8 months (95% CI 61–92), which was significantly higher than abatacept [37.5%, MST 37.1 months (95% CI 22–51; P = 0.01)]. Anti-TNF alpha therapy fell in the middle [50.0%, MST 63.5 months (95% CI 47–79)] but the difference was not significant. Nevertheless, seropositive RA patients did not show significantly different drug survival rates. Negative predictors of drug discontinuation were RF/ACPA positivity (HR 0.56) and sex male (HR 0.58), but treatment with abatacept (HR 1.88) or anti-TNF alpha (HR 1.79), no co-therapy with conventional DMARDs (HR 1.74), absence of bone erosions (HR 1.41) and higher HAQ (HR 1.58) were positive predictors. Conclusions To confirm these preliminary findings and to explore the hypothesis of a distinctive therapeutic algorithm in seronegative RA, prospective studies on larger cohorts are needed.