Abstract The effect of a mix of freeze dried Lactobacillus acidophilus plus Lactobacillus casei (2.3 × 10 8 colony forming units (cfu) kg −1 feed), or concentrates of Bacillus cereus (CICT-953) (10 9 cfu kg −1 feed), or Streptococcus faecium (CL-15) (1.5 × 10 9 cfu kg −1) on laying performance and mineral metabolism in White Leghorn hens was investigated in three 16-week experiments. A maize-barley (50/50) based diet (Experiment 1) or a barley based diet (Experiments 2 and 3) was used. The first experiment involved two treatments: a control diet and the same diet with L. acidophilus plus L. casei mixed culture. Supplementation improved hen day egg production ( P<0.01), feed conversion ratio ( P<0.01), egg weight ( P<0.05) and albumen quality ( P<0.05). Feed consumption and egg specific gravity were not influenced by the treatments. The second experiment also involved two treatments: an unsupplemented control diet and the same diet with B. cereus concentrate. Increased ( P<0.01) egg weight and albumen quality were observed in hens receiving the B. cereus culture. There were no significant differences in feed consumption, feed conversion ratio or egg specific gravity. The third experiment involved four dietary treatments: control diet; control diet with L. acidophilus plus L. casei mixed culture; control diet with B. cereus concentrate; or control diet with S. faecium concentrate. The L. acidophilus plus L. casei mix improved hen day egg production ( P<0.01), egg weight ( P<0.05), and albumen quality ( P<0.01). Feed consumption and feed conversion ratio were not affected by the addition of lactobacilli to the diet. Supplementation of B. cereus to the diet determined similar response to that indicated in Experiment 2. Birds receiving S. faecium produced greater egg mass ( P<0.01), egg weight ( P<0.01) and albumen quality ( P<0.01). Feed consumption, feed conversion ratio and egg specific gravity did not differ from the control group. Although calcium retention was not significantly affected by feeding the microorganisms cultures, it tended to improve by 2.4–4.2%. Addition of bacterial cultures to barley diet increased ( P<0.01) shell calcium. Plasma calcium, phosphorus, magnesium and zinc levels were similar to those of hens on the control diet. Feeding the S. faecium culture decreased ( P<0.05) serum copper. Laying hens fed an L. acidophilus plus L. casei mixed culture showed a decrease in serum iron ( P<0.01).