Lower quality ingredient use in broiler diets and maintenance of performance is a challenge. Under more challenging conditions, dietary supplementation with pro- (Pro) and post-biotics (Post) can elevate potential negative effects. The study aimed to assess the effects of a co-culture of Lactobacillus rhamnosus and L. formosensis-based Pro as well as the derived Post under two dietary conditions (standard [SD] and challenge [CD]), and determine their biological effects. Following a 2×3 randomised block design, day-old Ross male broilers (n=1368) were fed 1 of 2 diets (SD and CD) and as well as additives (Pro and Post) next to control diets from d1 to 42 with 6 pen replicates per treatment. Control SD and CD were formulated to meet broiler nutritional requirements and were iso-metabolizable energy, protein, digestible essential amino acids, calcium (Ca) and phosphorus (P), and fed from d1 onwards. The CD was formulated to be greater in non-starch polysaccharide content. Readout parameters included growth performance and tibia health, strength and composition at d35 as well as blood minerals at d21 and 35. Results were analysed by ANOVA and PERMANOVA, with body weight (BW) correction. Compared to SD, CD decreased BW at d42, while increasing 1-42d feed conversion ratio (FCR). CD also impaired bone health parameters (higher tibial dyschondroplasia (TD) and tibia head mineralization zone, lower tibia stiffness, dry matter (DM) and ash), thereby confirming the challenging effect of CD. Pro increased BW in CD but not in SD at d42, through increased feed intake, without affecting FCR. Post significantly increased BW in CD at d35 and numerically at d42. Pro increased plasma total Ca at d21 and 35 and P at d35, without affecting Ca/P and magnesium (Mg) at both ages. Post only increased plasma Mg at d21. Post but not Pro increased tibia stiffness and reduced TD score. Tibia DM and ash were not affected by Pro and Post. Tibia Ca and P content was only affected for Pro in SD with a reduction compared to the control SD. Tibia Mg was not affected by Pro and Post. Diet composition modulated the Pro and Post effects, with both improving broiler performance under “challenging” dietary conditions. Additionally, Pro and Post affected mineral metabolism, with potential beneficial effects of Post on TD and tibia strength.