Several studies have described variations in lactose content (LC) in dairy cows during udder quarter health disorder or negative energy balance (NEB). However, their joint effects on LC have never been described. This was the aim of a longitudinal observational study performed on 5 Quebec dairy farms using automatic milking systems. Quarter milk samples were collected every 14 d from 5 to 300 DIM. Quarter health status was described by combining SCC level (SCC- or SCC+: < or ≥100,000 cells/mL) and infectious status (Patho- or Patho+: presence or absence of pathogens on a milk culture). Cows with NEB in early lactation (DIM <70) were identified using milk β-hydroxybutyrate (BHB) content: 0.15 mM = BHB-; 0.15 - 0.19 mM = BHB+; > 0.19 mM = BHB++. A total of 14,505 quarter cisternal milk samples were collected from 380 lactating cows. The quarter LC was analyzed using a mixed linear regression model with the following fixed effects: quarter health status, parity, time interval between last milking and sampling, quarter milk yield (in kg/d), DIM, and herd. A random quarter intercept with a repeated measures correlation structure and a cow random intercept were also specified. The LC of SCC+ quarters was lower (-0.17 ± 0.013% pts) compared with LC of SCC- quarters for both primiparous and multiparous cows. Over the 162 bacterial species identified, only 8 species had a prevalence greater than 4.0%, and just 5 of them were associated with a reduction in LC: Staphylococcus aureus, Staph. chromogenes, Streptococcus dysgalactiae, Staph. epidermidis, and Staph. simulans. Cows identified as BHB+ and BHB++ in early lactation had a lower LC (-0.05 ± 0.019% and -0.13 ± 0.020% pts, respectively) compared with BHB- cows. For BHB++ cows, in both parity groups the decrease in LC (-0.20 ± 0.025% pts) was higher in SCC+ quarters compared with SCC- quarters. Moreover, the additive effect of the quarter health status and NEB on milk LC was greater with larger increases in BHB. Our findings highlight the necessity to jointly take into consideration both quarter health status and milk BHB concentration when using LC as a biomarker for NEB.